Technical Abstract:
A series of surveys were carried out in Brazil and Ecuador to collect endophytic fungi for use as biological control agents. The isolates collected consisted of a range of fungi, including several known and new species of Trichoderma. The endophytic habit of these Trichoderma isolates within the tissues of cocoa (Theobroma cacao) both above and below ground was confirmed. Cacao seedlings were inoculated using agar plugs by a variety of methods including inoculation on water agar plates, inoculation of seedlings at the soil line or between the cotyledons, or by planting cacao seed in pre-colonized soil. The isolates were shown to differ in their abilities to colonize, depending on the cocoa tissue being studied (root, stems, cotyledons, leaves, xylem, plumules, or bark) and the method of inoculation. Changes in gene expression in response to colonization were identified in cocoa seedlings using differential display techniques. From 166 differential display bands that were cloned and sequenced, 115 unique ESTs were identified. Putative functions for 58 of the 115 unique ESTs were determined based on sequence homology. A 142 member macro-array was developed consisting of the 115 endophyte associated ESTs plus 27 previously studied ESTs associated with plant defense in cacao. After macro-array analysis, the expression of differentially expressed cacao and fungal ESTs was further studied using real-time PCR techniques. Endophyte associated gene expression will be used to monitor the impact and persistence of Trichoderma species when applied to cacao in the field.